A fundamental challenge in neuroscience and regenerative medicine is to understand how specific neuron subtypes arise in correct number, at defined times, and target to precise locations. Programs enacted to coordinate such events have begun to be defined, for example, through studies of early-stage spinal neural tube; in part due to physical and molecular accessibility, and relatively simple anatomy--newly born neurons typically move short distances radially, maintaining originating DV/AP relationships. Less clear is how such events are regulated in germinal zones that distribute neuron cohorts to distant and varied locations through tangential migration modes, and which typically do so at later developmental stages. Yet such germinal zones contribute substantially to neuron diversity in the brain and must do so in novel ways given their unique properties and the distinct milieu of late-gestation neural tissue. This proposal seeks to fill key aspects of this knowledge gap through studies of the hindbrain (lower) rhombic lip (LRL), a late-acting and essential germinal zone now accessible by molecular genetic means. Diverse and functionally critical brainstem cell types arise from the LRL, many subject to developmental and degenerative disorders. We provide evidence that (1) this diversity in produced cell types is reflected in a precisely defined molecular sub-regionalization of the LRL - the LRL is not homogeneous, but rather comprised of DV and AP gene expression microdomains predictive of progeny neuron identity; (2) the transcription factor Pax6 modulates aspects of this molecular sub-regionalization, ensuring that correct proportions of descendant lineages are produced; and (3) the archetypal ventralizing morphogen, Sonic hedgehog (Shh), is expressed in the dorsally situated hindbrain choroid plexus epithelium, a known dorsal organizing center and itself a LRL lineage. Such co-opting of previously excluded patterning molecules, such as Pax6 and Shh, may represent a general strategy by which a late-acting germinal zone may continue generating unique progenitor cell states and fates. Our goal is to two-fold: Extend our LRL fate map by linking highly resolved DV:AP coordinates to later-generated cell fates (Aim 1); place on this map the action of molecules which regulate this molecular organization and thus production of specific cell types; for example, we will explore novel roles for Pax6 and Shh (Aims 2 & 3). [unreadable] [unreadable] [unreadable]